Detection device for detecting biological microparticles such as bacteria, viruses, spores, pollen or biological toxins, and detection method

ABSTRACT

A device for the detection of micro particles that can be marked by probes or antibodies capable of being detected by radiation has a filter, a supply system, and a detection system. Fluid to be examined is passed over a filter to filter out the micro particles and to perform the marking steps by supplying corresponding marking substances to the filter.

This application is a divisional of prior application Ser. No.12/598,483, filed on Jan. 28, 2010, which is a U.S. National stageapplication that claims priority under 35 U.S.C. §119(a) to GermanPatent Application No. 10 2007 021 387.7, filed in Germany on May 4,2007, the entire contents of U.S. application Ser. No. 12/598,483 andGerman Patent Application No. 10 2007 021 387.7 are hereby incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a detection device for detecting biologicalmicro particles such as for example living or dead bacteria, viruses,spores, pollen or biological toxins that can be marked using probescapable of being detected by radiation, in particular a detection devicefor detecting living bacteria, viruses or biological toxins by means offluorescent nucleic acid probes or proteins as probes. The inventionalso relates to a detection method for detecting biological microparticles such as for example living bacteria, viruses, spores, pollenand/or biological toxins in a fluid.

2. Background Information

Previous techniques for detecting living bacteria require atime-consuming step of cultivation of the micro organisms. Thisconventional technique of detection of living cells or cell numberdetermination of living cells requires an enormous amount of time andcan be performed only in suitable biological laboratories (S1 to S4laboratories). An alternative technique for the detection of livingbacteria is the in situ hybridization. The in situ hybridizationtechnique is a standard technique already frequently applied inmolecular biology. There have been numerous publications on thistechnique.

Concerning bacteria detection, the company of Vermicon AG, Munich,offers ready-to-use detection kits, i.e. ensembles of chemicals, fordifferent bacterial strain.

Previous detection methods including in situ hybridization are describedin more detail in WO 01/68900 A2, WO 02/101089 A2, DE 103 07 732 A1, WO2005/031004 A2, U.S. Pat. No. 6,844,157B2, US 2005/064444 A1,US/20050202477 A1, US 2005/0202476 A1 as well as US 2005/0136446 A1. Forfurther details it is explicitly referred to these prior art documents.

Presently however, the in situ hybridization is possible in the practiceonly by using a very expensive and highly sensitive fluorescencemicroscope.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod allowing rapid, reliable and uncomplicated automatic detectionespecially of living bacteria, but also of other micro particles andmicroorganisms, in particular viruses or biological toxins, that can bemarked using special probes.

This object is achieved by a detection device comprising the features ofa first aspect of the present invention and by a detection methodcomprising the steps of a twenty-fourth aspect of the present invention.

Advantageous embodiments of the invention form the subject of otheraspects. A beneficial use forms the subject of another aspect.

Advantageous embodiments of the invention enable in particular anautomatic live detection of pathogenic bacteria in drinking water andair.

The invention particularly provides a device for rapid and preferablyautomatic detection of living bacteria, without the aid of afluorescence microscope.

The measuring results are especially suitable for being used to obtain acorrelation to CFU (colony forming units), with the aim of providing analternative to the approved time-consuming cultivation method (whichprovides the CFU value).

According to the invention, a filter is provided suitable for filteringout the micro particles to be detected. The substances or chemicalsserving in the various marking steps to mark the micro particles for thepurpose of detection are provided for instance by a supply system andare also passed to and particularly over the filter. By means of adetection system, the radiation for detecting the probes can be detectedand thus also the micro particles.

Especially for the detection of living bacteria a micromechanical filteris used for example. In such a case, a supply system delivers especiallythe chemicals to be used in the individual steps of an in situhybridization, for instance from detection kits available on the marketfor detecting particular bacteria.

The supply system can be preferably automatically controlled via valvesand/or pumps, so that the detection method can be performed fullyautomatically.

Preferably, the filter can be used several times by being regeneratedand/or conditioned using suitable chemicals. This is enabled bycorrespondingly controlled pumps and valves.

In particular, the supply system can be a part of a fluidic system whichdoes not only supply chemicals that are used for marking and/or for thelater washing and cleaning of the device, but which is also capable ofdelivering fluids to be measured to the filter. The measurement ofradiation is adapted to the respective probes that are used.

In order for the probes to emit radiation, fluorescent colorants or forinstance also Raman labels, quantum dots or the like can be used. Ifprobes marked with a fluorescent colorant are used, as this is knownalso in the in situ hybridization, a light source for exciting thefluorescent colorant is preferably employed, for example a laser.

As a radiation measuring device for the measurement of radiationcorrelated to the probes preferably a light detector is used which issensitive to a particular fluorescence radiation.

The light detector may include for example a photo multiplier, in orderto thus detect the intensity of the fluorescent light. Alternatively oradditionally a space-resolving light detector, for example atwo-dimensional space-resolving light detector can be used. Such adetector is formed for example by a two-dimensional CCD array. Such aspace-resolving sensor is capable of determining light pulses and theposition of their origin on the filter. Due to the higher resolution anddue to the individual sensor elements an evaluation unit, which ispreferably controlled by software, can be connected for counting e.g.the light spots and thus the probes and thus the micro particles.

For filtering out living bacteria, a micromechanical filter element canbe used for example. Preferably employed are micromechanical filtershaving a pore size clearly below the size of the micro particles to befiltered out. A micromechanical filter for instance has a pore size ofless than 0.8 μm, for example approx 0.45 μm, so that bacteria havingtypical dimensions of 1 μm are securely retained.

Other interesting micro particles, such as for example viruses orbiological toxins, partly have smaller dimensions. For filtering thesemicro particles, a micromechanical filter having a small pore size or adifferent filter, particularly a nitrocellulose filter, can be used. Forthis purpose, a micromechanical filter or a different porous body as asupport are covered with nitrocellulose. The nitrocellulose membrane canbe provided for instance by a web capable of being moved for theexchange of the effective nitrocellulose membrane, so that a fresh webpiece can be used as a filter for new measurements.

For the determination particularly of viruses or biological toxins,proteins detectible by radiation such as antibodies marked with afluorescent colorant or marked nucleic acid probes can be used.

For the detection of living bacteria, correspondingly marked DNA probesare preferably employed which attach to the mRNA that is only providedby living bacteria having a sufficient lifetime.

Additionally to this probe marking employed in the in situ hybridizationalso a PCR module can be used to alternatively or additionally perform aPCR detection process.

In the PCR method, which is also employed in criminology, pieces of DNAare multiplied and thereafter detected.

If the concentration of micro particles in liquids is to be determined,for example the number of living bacteria in drinking water, this liquidcan be directly supplied to the detection device and passed over thefilter. If a concentration of micro particles in gaseous media is to bedetermined, for example in the air, these micro particles can be firstcollected in a liquid, for example in an upstream collecting device,e.g. a bio sampler, and this liquid can subsequently be transferred tothe detection system, or the particles can be directly collected fromthe air by passing the air through the filter.

Advantages of the invention and/or advantages of beneficial embodimentsof the invention are as follows:

-   -   A rapid, uncomplicated and sensitive automatic detection of        living bacteria using the detection device is possible at any        location.    -   Time-consuming steps of cultivating bacteria in a biological        laboratory can be omitted.    -   A mobile detection of living cells is possible. This allows for        example rapid testing of drinking water in a vehicle or aircraft        or spacecraft or, in connection with a robot, for civil and/or        military purposes.    -   The device and the method enable for example rapid examination        of suspect liquids, in order to prevent biological attacks, and        thus contribute to public safety.    -   In medicine for example, the device and the method allow a rapid        diagnose of bacterial diseases, without examination through the        microscope.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to theattached drawings, wherein it is shown by:

FIG. 1 a DNA sequence for explaining the basics of in situhybridization;

FIG. 2 a schematic illustration of the principle of in situhybridization;

FIG. 3 a schematic illustration of a rapid detection device fordetecting living bacteria in drinking water;

FIG. 4 a schematic illustration of a construction example of a rapiddetection device for detecting living bacteria in fluids using a fluidicsystem with pumps and valves as well as a detection chamber with laserand photo multiplier;

FIG. 5 a schematic drawing of the detection chamber from above, saidchamber including a micro mechanical filter;

FIG. 6 a schematic illustration of the detection chamber and of adetector of a further embodiment of a detection device;

FIG. 7 a schematic illustration of the detection chamber and ofdetectors of a further embodiment of the detection device in which acombination of in situ hybridization and PCR detection can be performed;

FIG. 8 a schematic illustration of the detection chamber according to afurther embodiment; and

FIG. 9 a schematic illustration for explaining the use of the detectiondevice according to FIG. 8, for detecting viruses and biological toxins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A method and a device for the detection of micro particles that can bemarked by probes capable of being detected by radiation will be moreclearly described in the following with reference to the drawings. Thedevice and the method are particularly suited for the detection ofliving bacteria by means of nucleic acid probes or of viruses orbiological toxins by means of proteins including antibodies for theprobes. Before explaining the device and the method, the general methodsfor marking such micro particles by means of such probes will be moreclearly explained.

Drinking water contains different living and dead bacteria. A partthereof can be pathogenic. Only the living bacteria are relevant,because these alone multiply and may constitute a health risk.

But many conventional detection methods based upon antibodies or PCR areincapable of differentiating between living and dead bacteria. In thefollowing, a device and a method will be described which in fact allowthe detection only of the living and hence the relevant bacteria.

Previous detection methods for detecting living bacteria require atime-consuming step of cultivating micro organisms. For this purpose,the bacterial samples are streaked out on special nutrient media and areincubated (breeded) at a particular temperature for a particular time.Depending on the bacterial species, this cultivation step may continuefor several days. After the end of the incubation period, the colonyforming unit (CFU), the number of bacteria colonies that have formed, isdetermined. This conventional technique of living detection ordetermination of the number of living cells is extremely time-consumingand up to present there is no possibility of a rapid and uncomplicateddetection of living bacteria.

An alternative technique for the detection of living bacteria is in situhybridization. But presently this analyzing method can be carried outonly in a very expensive and highly sensitive fluorescence microscope.Mobile field tests are practically impossible and qualified laboratorypersonnel is required for operating the microscope.

For the detection of microorganisms additional detection methods arefrequently applied, for example polymerase chain reaction (PCR) orenzyme-linked immuno sorbent assay (ELISA). But these methodsexclusively determine the number of the living and dead cells, hence thetotal number of cells of a sample. The determination of the number ofliving cells is not possible with these methods.

2. Methods for Bacteria Detection

A) PCR

The PCR method (polymerase chain reaction) is a method in which smallestamounts of a DNA section can be multiplied in a chain reaction(amplification). Today, the PCR method is frequently used for variousproofs by way of particular DNA sequences, for example:

-   -   in criminology or in paternity proof    -   in microbiology, for the detection of living and dead bacteria        (total cell number)    -   in medical diagnose, for the proof of virus DNA in the blood, or    -   in evolution biology, for tracing family relations and lines of        ancestry.

For PCR detection, two short pieces of DNA (primer) must be presentmatching the searched DNA strand. The chain reaction initiated by themperforms up to 30 cycles, the amount of DNA being doubled at each cycle.Due to the detection via genotype, dead and living bacteria aredetected. A detection exclusively of living cells is not possible withthe PCR method.

B) ELISA

ELISA (enzyme-linked immuno sorbent assay) is a wide-spread method ofdetecting individual proteins (antigen) using the mechanisms of theimmune system: If a substance is recognized by the immune system as aforeign substance, the immune system produces antibodies docking to theforeign molecule and thus marking the molecule. This so-calledantibody-antigen interaction is utilized for the ELISA test.

If a particular protein has to be detected, the matching antibodies mustbe known and must have been prepared in advance using variousgen-technological or cell-biological methods. If the searched protein ispresent in a sample, the protein will be bound by the antibodies appliedto a substrate. After the antigen-antibody interaction a reactioncontrolled by enzymes is induced which results in a visible inkprecipitate. ELISA tests are very common today in medical diagnose. Butthey are also used in many other fields, if individual proteins have tobe detected. In the case of bacteria detection, the antibody recognizessurface proteins specific to bacteria. Both the living and dead bacteria(total cell number) are detected.

C) In Situ Hybridization

Reference is made in the following to FIG. 1. A DNA sequence 10 may beimagined as a “zipper”. The “teeth” of this zipper are the bases adenine(A), cytosine (C), guanine (G) and thymine (T). The informationcontained in the DNA is encoded along the “zipper” in the order of thesefour letters. Opposite “teeth” always only form either AT or GC pairs.The sequence ACGCT for example has the letter sequence TGCGA as acomplementary vis-à-vis.

During the hybridization, the “zipper” is opened by heating, so thatsingle strands are exposed. Short pieces of DNA, which are alsosingle-stranded pieces of DNA and which are used as probes, are nowsupposed to find their matching counterpart on this long single strand.This is the point where the zipper closes again after cooling.

This can be made visible by means of markings on the probe (e.g. bymeans of a fluorescent colorant 11, see FIG. 2). In this way it ispossible to find out whether or not specific sequences, representing forexample particular marker gens, are present in the examined DNA (seeitem A: PCR).

In the case of in situ hybridization, which is explained in more detailin the following with reference to FIG. 2, the fluorescence-markedprobes 12 do not associate with the DNA, but instead with the mRNA 14,the so-called “copy” of the DNA. The mRNA 14 is a kind of copy of theDNA. From the information transcribed into the mRNA, proteins are formed(protein biosynthesis).

In dead bacteria this mRNA 14 has an extremely short lifetime(microseconds) and it is instantly degraded after the dying of thebacteria.

With the in situ hybridization method the mRNA of living cells ispreserved (technical expression: “fixed”) and then detected, i.e.exclusively the living bacteria are detected (number of living cells),since dead bacteria do no longer contain mRNA.

Since in this method DNA binds to (m)RNA, one speaks of hybridization.

Even a combination of in situ hybridization and PCR is possible. In thiscase, the DNA of the bacteria which is also fixed is used foramplification. This method, which is called in situ PCR, is employed forinstance for the parallel detection of viruses in the sample to beexamined.

In the meantime, the in situ hybridization method has developed into astandard method which is already frequently used in molecular biology.For further details concerning the individual steps of the in situhybridization and of the other detection methods herein presented it isexplicitly referred to WO 01/68900, WO 02/101089 A2, DE 103 07 732 A1,WO 2005/031004, U.S. Pat. No. 6,844,157 A1, US 2005/064444 A, US2005/0202477 A1, US 2005/202476 A1, US 2005/0136446 A1.

Ready-to-use detection kits with ensembles of chemicals are available onthe market, e.g. from the company of Vermicon AG, Emmy-Noether-Strasse2, D-80992 Munich, Germany. However, the detection kits which arecurrently available on the market require a fluorescence microscope, andthe chemicals must be professionally used by qualified personnel.

Embodiments

On the other hand, a detection device 20 enabling a fully automateddetection of living bacteria without the use of a microscope isillustrated in FIG. 3. The detection device 20 comprises a detectionchamber 22 in which a filter 24 is arranged for filtering out the microparticles or microorganisms to be detected from the fluid to beexamined.

For the detection of living bacteria the filter 24 is constructed as amicromechanical filter and thus includes a micromechanical filteringelement 26. The micromechanical filter and its filtering element forexample have a structure which is more clearly described and illustratedin the German patent application DE 10 2006 026 559.9, which is not aprior publication. For further details reference is made explicitly toDE 10 2006 026 559, the contents of which is incorporated herein byreference.

The detection device 20 also includes a fluidic system 28 for passingthe fluid, e.g. drinking water, to be examined for the presence ofliving bacteria 32, to and through the filter 24.

The fluidic system 28 comprises one or more pumps 34, 35 and alsoseveral motor valves A, B, C, D. The pumps 34, 35 and the valves A, B,C, D can be automatically controlled by a control unit (not furtherillustrated) connected to it, e.g. a computer including correspondingsoftware.

Moreover, a part of the fluidic system 28 serves as a supply system 36adapted for supplying chemicals for marking the bacteria 32 or othermicroorganisms or micro particles to be detected by means of probescapable of being detected by radiation, for example fluorescence-markedprobes 12, to the filter 24. Another part of the fluidic system 28serves as a discharge system 38 for discharging substances and samplesfrom the detection chamber 22.

More precisely, the supply system 36 of the fluidic system 28 comprisesa motor valve A having an outlet A1 and several inlets A2 to A9. Thefollowing table 1 shows the configuration of the inlets for a practicalexample.

TABLE 1 Configuration of the inlets of the motor valve A in a practicalexample. Valve inlet A2 A3 A4 A5 A6 A7 A8 A9 20% H₂O reserve PBS sampleof antibody wash 0.5M EtoH (inlet is drinking buffer NaOH free) water

Two pumps 34 and 35, which are connected in parallel and which have adifferent pumping capacity, connect to the outlet A1. A first pump 34works for example in a working range of 6 to 70 ml/min, and a secondpump 35 works in a working range of 0.1 to 7 ml/min. Depending on thecontrol and on the switching of the pumps 34, 35, precisely dosedamounts of fluids delivered from the motor valve A can pumped towardsthe detection chamber 22.

Following the pumps 34, 35 is a 3/2 directional valve B enabling theoutput of the pumps 34, 35 being selectively directed to one of the twosides of the filter 24. To this end, the detection chamber 22 issubdivided into two sub chambers 42 and 44 by a partition 40, and thesub chambers are interconnected trough the filter 24. While the inlet B1of the valve B is connected to the outlet of the pumps 34, 35, an outletB2 of the valve B is connected to the first sub chamber 42, and a secondoutlet B3 of the valve B is connected to the second sub chamber 44.Depending on whether the first or second sub chambers 42 or 44 arecharged, the filter 24 can be charged with corresponding liquids,cleaned and/or conditioned. Corresponding flows can also be achieved viathe discharge system 38 in which a first inlet C1 of a 3/2 directionalvalve C is connected to the first sub chamber 42 and a second inlet C3is connected to the second sub chamber 44. The 3/2 directional valve Cselectively allows the first inlet C1 or the second inlet C3 beingswitched to an outlet C2. The outlet C2 is connected in turn to an inletD1 of an additional 3/2 directional valve D. This inlet D1 can beselectively switched by the valve D to a drain 46 for dischargingantibodies or to a manifold 48 for collecting antibodies.

That means that by the arrangement of openings for supplying liquid onboth sides of the filter and of openings for discharging liquid on bothsides of the filter and also by the arrangement of the valves B and C, aflow through the filter 24 in both directions is achieved. By theswitchable valve A, which is connected to two or more liquid tanks,chemicals for marking, cleaning or conditioning can flow through thefilter.

For the detection of the bacteria 32 marked with the hybridizationprobes or with fluorescence-marked proteins or antibodies, a radiationmeasuring device 50 is connected to the detection chamber 22.

To detect for instance bacteria 32 which are marked usingfluorescence-marked probes 12, a light source in the form of a laser 52is required which emits radiation suitable for exciting the fluorescentcolorant 11, for example light having a wavelength of 405 nm. Thislimitation of the wavelength can be further improved by an opticalfilter connected downstream of the light source.

In the illustrated embodiment the radiation measuring device 50 includesa light detector 54 for detecting the radiation which is used here,namely fluorescence light 56 from the bacteria 32. For a more precisedetection of such radiation, the radiation measuring device 50 furtherincludes an optical filtering element 58 which only allows the radiationto be measured, in the present case the fluorescence light 56 to bemeasured, e.g. fluorescence light at a wavelength 455 nm, to passthrough.

For the connection of the radiation measuring device 50, the detectionchamber 22 is closed on one side with a quartz glass 60. Excitation bymeans of the laser 52 and light detection by means of the light detector54 take place through this quartz glass 60.

FIG. 4 shows a general drawing of a more special first embodiment of thedetection device 20, wherein the detection chamber 22 and the fluidicsystem 28 are only schematically indicated. In the embodimentillustrated in FIG. 4, the light detector 54 includes a device formeasuring the intensity of light, in the present case a photo multiplier62. In the present case a fluidic system 28 including only a single pump34 and a simplified discharge system 38 is used.

The detection device 20 shown in the FIGS. 3 and 4 allows a fullyautomatic detection of living microorganisms using the method of in situhybridization. Within this system, living bacteria 32 are rapidly andreliably detected for the first time using the in situ hybridizationmethod. The core of the device or of the detection device 20 is thedetection chamber 22 containing the micromechanical filter 26. Throughthe fluidic system 28 (pumps 34, 35, passages or hoses and valves A, B,C, D) connected to the detection chamber 22, the sample to be examined(e.g. water 30 containing bacteria 32) is pumped over the filteringelement 26.

Since the micromechanical filtering element 26 has a pore size of e.g.0.45 μm and since the bacteria 32 have a diameter of e.g. 1 μm, thebacteria 32 will accumulate on the filter surface.

After their accumulation, the bacteria 32 are prepared for the actualdetection. To this end, the chemicals required for the in situhybridization are pumped over the filtering element 26 with the aid ofthe fluidic system 28, especially with the aid of the supply system 36.

The actual detection is performed using a light source, e.g. the laser52, which excites the fluorescent colorant 11 with which the DNA probe12 has been marked. The fluorescence light 56 emitted as a result of theexcited fluorescent colorant 11 is subsequently detected by means of thephoto multiplier 62 (PMT). Special optical filters—in the present casethe optical filtering element 58—ensure that the photo multiplier isreached exclusively by fluorescence light 56 emitted from the markedbacteria 32.

FIG. 5 is a schematic illustration of the detection chamber 22 includingthe filter 24 as viewed from above. The grid that can be recognizedrepresents the micromechanical filtering element 20. Also shown areconnections 64 and 66 to the supply system 36 respectively the dischargesystem 38 of the fluidic system 28.

Target organisms for a living detection are for example pathogenicgerms. Due to the base sequence of the fluorescence-marked probe 12, itis not only possible to quite specifically detect a pathogenic bacterialspecies (e.g. E. coli), but it is also possible to detect bacteria on ahigher taxonomic level. Hence, in the case of E. Coli, it might bepossible that all coli species in a sample are detected.

By designing the base sequence of the DNA probe 12 to be moreunspecific, it may become possible to detect on an even higher taxonomiclevel, e.g. to detect many enterobacteria.

Even a simultaneous detection of different bacterial species ispossible. To this end, a mixture of differently marked probes 12 can beemployed, using a specific fluorescent colorant 11 for the respectivespecies of bacteria. In this way, it can be determined which bacteria 32are present in the concentration, if excitation takes place at differentwavelengths.

FIG. 6 schematically shows the detection chamber 22 including the filter24, and the light detector 54 of a further embodiment of the detectiondevice 20. In this embodiment according to FIG. 6, the light detector 54does not include a photo multiplier, but a space-resolving detector,namely in the present case a two-dimensional space-resolving detector inthe form of a two-dimensional CCD array that is connected to anevaluation unit, for example a computer system 70.

If the detection is not made through a photo multiplier 62, buttwo-dimensionally by means of a two-dimensional space-resolvingdetector, e.g. a CCD array 68, plane above the filter 24, the positionof the individual bacteria 32 on the filter 24 can be determined. Thepresentation or resolution on the CCD array can be improved by opticscomprised of optical lenses.

By using suitable software, the individual bacteria 32 can be counted.This allows the actual number of the bacteria 32 being determined moreprecisely than by measuring with a photo multiplier 62, because in thelatter only the total light intensity is measured.

From the light intensity obtained by the photo multiplier 62 the numberof bacteria 32 that had been present is derived through correspondingcalibration. Compared thereto, the detection system illustrated in FIG.6 is capable of immediately outputting the number of bacteria.

The inaccuracy of a measurement through light intensity is the higherthe more different is the concentration of mRNA 14 in different bacteria32.

To detect bacteria in the air or in other gaseous media using theabove-described embodiments of the detection device 20, these can becollected for example automatically through a suitable collecting devicein a liquid and supplied together with the liquid to the detectiondevice 20, for example through the connection A6. Suitable detectiondevices are available on the market for example under the nameBiosampler from the company of SKC Inc., Eighty Four, Pa., USA.

Alternatively, the air may be directly passed through the filter, inorder to accumulate the particles on the filter.

The detection method that can be carried out with the detection device20 is capable of replacing the current certified cultivation methods forCFU determination. To this end, the probes 12 which are used are soselected that the bacteria 32 detected with these probes are similarlyrepresentative as the bacteria detected by the conventional CFUdetermination.

FIG. 7 still shows in a schematic representation the detection chamber22 including the filter 24 and the radiation measuring device 50 of afurther embodiment of the detection device 20.

The embodiment according to FIG. 7 includes an additional module 72—herein the second sub chamber 44—for PCR detection of DNA sequences. Theadditional module 72 is for example a device for carrying out a realtime PCR. Such devices are available on the market, for example from thecompany of Fluidikm Corp., South San Francisco, USA.

The additional module 72, which is employed in addition to the actualdetection device 20, enables a combination of in situ hybridization andPCR. A so-called in situ PCR can be used for example for a subsequentdetection of viruses. The actual detection could be made using themethod of real time PCR. Thereby, reporter molecules are released duringthe amplification and due to their fluorescence the reporter moleculescan be detected by a photo multiplier.

To this end, the additional module 72 is equipped with a separateradiation measuring device for measuring fluorescence light or itutilizes the correspondingly constructed radiation measuring device 50of the detection device 20.

The embodiment illustrated in FIG. 7 particularly enables the specialembodiment of the detection method more clearly described in thefollowing being carried out. A combination of in situ hybridization andPCR are performed. In the in situ hybridization DNA probes 12 are addedafter fixing the bacteria 32. The non-bound DNA probes 12 willsubsequently be washed away. If the bound DNA probes 12 are now resolvedagain by the mRNA 14 and amplified by PCR, living bacteria detection canthus be made which even allows detecting extremely small bacteriaconcentrations.

Hence, the PCR method here not only serves for determining the totalnumber (of dead and living bacteria). On the contrary, the DNA probespreviously bound to the mRNA 14, which are only present in livingbacteria having a sufficient life time, and multiplied many times by thePCR are detected.

FIG. 8 shows the detection chamber 22 and the filter 24 of a furtherembodiment of the detection device 20 which is also capable ofautomatically detecting viruses and biological toxins. To this end, amembrane, particularly a nitrocellulose membrane 74, is provided. Thenitrocellulose layer 74 can be present for example on any permeable bodyserving as a support. In the embodiment of the detection devices 20illustrated in the FIGS. 8 and 9 the nitrocellulose membrane 74 isprovided as a layer over the micromechanical filtering element 26.

FIG. 9 illustrates the principle of the filtration of viruses 76 andbiological toxins 78 by means of the nitrocellulose as well as thedetection by means of fluorescence-marked antibodies as probes which areused for the detection in this detection method.

Accordingly, the FIGS. 8 and 9 more clearly explain a furtherapplication of the rapid detection device 20 for the detection ofpathogenic viruses 76 and biological toxins 78.

Viruses 76 are small particles (25˜500 nm) consisting of a protein shelland—depending on the species of the virus—a RNA or DNA genome. They donot have a metabolism of their own, but propagate exclusively throughliving cells. Known examples are influenza viruses, ebola viruses orsmallpox viruses.

Biological toxins 78 are extremely stable proteins which are mostlyformed from bacteria, unicellular organisms or plants as metabolicproducts.

These toxins are frequently released into the surrounding medium wherethey become enriched in high concentrations. In the military fieldbiological toxins can be used as biological weapons.

Known representatives of biological toxins are botulinumtoxin (botox)and ricin that can be obtained from the seeds of the castor plant.

Due to their small size, viruses 76 and biological toxins 78 canaccumulate only to a limited extent on micromechanical filters. Bycovering the micromechanical filtering element 26 or a differentpermeable body, which serves as a support, with a suitable absorbingmaterial, especially nitrocellulose 74, viruses 76 and biological toxins78 may nevertheless be successfully filtered.

Nitrocellulose exhibits great affinity towards proteins and nucleicacids. Due to their surface condition, proteins are irreversibly boundto the nitrocellulose membrane 74. In this way, whole viruses with theirsurface proteins can be fixedly bound to the nitrocellulose membrane 74.

Binding proteins and whole viruses to nitrocellulose is a widely spreadmethod in the field of biological research and is described in numerouspublications (“Westernblot” or “Dotblot”).

After the viruses 76 have become adhered to the diaphragm 74, thepositions of the membrane which are still unoccupied can be filled upwith particular proteins (e.g. BSA bovine serum albumin) 82. Thisprocess is called blocking the membrane. After the blocking the actualdetection with specific antibodies 80, which have been marked withfluorescent colorant 11, takes place.

Due to this special property of the nitrocellulose it is possible tosuck the sample and all the liquids required for detection through themembrane 74.

Should sucking through the membrane 74 be restricted for some reasons oreven be impossible, the membrane 74 could also be superficially washedusing the detection device. This would merely require a differentswitching sequence of the 3/2 directional valves B, C which arepositioned next to the detection chamber 22 on the left and right sidesthereof (FIG. 4).

All steps of all of the above-described detection methods can be carriedout fully automatically with the present device—the detection device20—that has been developed, so that analyses are possible also withoutspecially trained personnel.

Other than for the examination of liquids a device equipped withnitrocellulose could also be used for the examination of air. If viruses76 or toxins 78 present in air shall be detected, the same automaticprogram as for liquids can be used, with air or any other gaseous mediumbeing pumped through the filter.

For making numerous measurements one after the other, the membrane 74can be provided in the form of an endless roll of nitrocellulose, a unitof which is paid off after each measurement, thus ensuring that thefilter 24 is fresh prior to each measurement.

What is claimed is:
 1. A detection method to detect biological microparticles in a sample, comprising: providing a fluid containing thebiological micro particles at a positive pressure into a detectionchamber on a first side of a filter enclosed in the detection chamberand applying a negative pressure to the detection chamber on a secondside of the filter, opposite to the first side, to draw the fluidthrough the filter such that the filter retains the biological microparticles adhering to the first side of the filter; providing a chemicalfluid into the detection chamber by operating a first valve toselectively connect one of a plurality of sources of chemicals to anoutlet connection to supply to the outlet connection the chemical fluidfrom one of the sources, operating first and second pumps, each with adifferent pumping capacity, to provide the chemical fluid from theoutlet connection to a valve inlet, and operating a second valve toprovide the chemical fluid from the valve inlet at a positive pressureinto the detection chamber on the first side of the filter, and applyinga negative pressure to the detection chamber on the second side of thefilter to draw the chemical fluid through the filter to chemically markthe biological micro particles adhering to the first side of the filterby specific probes configured to be detected by radiation while thefilter remains enclosed in the detection chamber and to discharge thechemical fluid that has passed through the filter out of a dischargesystem; performing a polymerase chain reaction (PCR) on the biologicalmicro particles adhering to the first side of the filter that have beenmarked by the specific probes; emitting excitation light into thedetection chamber to irradiate the probes with the excitation light toexcite the probes to produce emitted light while the biological microparticles adhere to the filter that remains enclosed in the detectionchamber; detecting the emitted light while the filter remains enclosedin the detection chamber to detect the radiation present in the emittedlight that correlates to the probes; and detecting the presence of thebiological micro particles in the sample while the filter remainsenclosed in the detection chamber based on the emitted light.
 2. Thedetection method according to claim 1, wherein the biological microparticles adhering to the filter include living bacteria; and thechemically marking of the biological micro particles includes subjectingthe bacteria to an in situ hybridization.
 3. The detection methodaccording to claim 1, wherein the chemically marking the biologicalmicro particles includes marking the biological micro particles with DNAprobes provided with a fluorescent colorant; and the fluorescentcolorant is excited by the excitation light and the emitted light isemitted due to excitation of the fluorescent colorant and is detected bya light detector during the detecting of the radiation correlating tothe probes.
 4. The detection method according to claim 3, wherein thedetecting of the radiation includes detecting a light intensity by thelight detector.
 5. The detection method according to claim 4, whereinthe detecting of the radiation includes detecting the light intensity bya photo multiplier which is included in the light detector.
 6. Thedetection method according to claim 3, wherein the detecting of theradiation includes detecting the radiation or light pulses and aposition of their origin on the filter using a space-resolving lightdetector as the light detector; and the detection method furtherincludes counting the light pulses using a data processing system. 7.The detection method according to claim 1, further comprising chemicallymarking the biological micro particles with unspecific DNA probes thatmatch with nucleic acids or mRNA of different bacterial species;irradiating the unspecific DNA probes with the excitation light toexcite the unspecific DNA probes to produce additional emitted light;detecting additional radiation present in the additional emitted lightthat correlates to the unspecific DNA probes; and determining a totalconcentration of bacteria of a plurality of bacterial species in thebiological micro particles based on the detected additional radiation.8. The detection method according to claim 1, further comprisingchemically marking the biological micro particles with differentlymarked probes; irradiating the differently marked probes with theexcitation light to excite the differently marked probes to producedifferent emitted light; detecting additional radiation present in thedifferent emitted light that correlates to the differently markedprobes; and determining a presence of several different bacterialspecies in the biological micro particles at the same time based on thedetected additional radiation.
 9. The detection method according toclaim 1, further comprising collecting the biological micro particlesfrom the air or from other gaseous media in a liquid; and the passing ofthe fluid containing the biological micro particles through the filterincludes passing the liquid containing the biological micro particlestogether with the fluid over the filter.
 10. The detection methodaccording to claim 1, wherein the detecting for the occurrence of thePCR of the biological micro particles includes detecting the emittedlight while the PCR occurs as an in situ PCR process for detection ofviruses.
 11. The detection method according to claim 1, wherein thedetecting for the occurrence of the PCR of the biological microparticles includes detecting for reporter molecules that are releasedduring amplification by detecting their fluorescence using a photomultiplier.
 12. The detection method according to claim 1, wherein thechemically marking of the biological micro particles includes chemicallymarking the biological micro particles with bacteria DNA probes thatbind to mRNA; washing away non-bound DNA probes from the biologicalmicro particles; and performing the PCR to amplify the DNA probes thatare bound to the mRNA of the biological micro particles.
 13. Thedetection method according to claim 1, wherein the filter includes atleast one of a micromechanical filtering element and a nitrocellulosemembrane; and the passing of the fluid containing the biological microparticles through the filter includes passing the fluid containing thebiological micro particles through the at least one of themicromechanical filtering element and the nitrocellulose membrane. 14.The detection method according to claim 13, wherein the filter includesthe nitrocellulose membrane; and the chemically marking of thebiological micro particles includes filling up with proteins thosepositions of the nitrocellulose membrane that are unoccupied.
 15. Thedetection method according to claim 13, wherein the filter includes thenitrocellulose membrane; and the chemically marking of the biologicalmicro particles includes marking the biological micro particles adheringin the nitrocellulose membrane by the specific probes which includeantibodies or marked probes that are configured to be detected based onthe radiation.
 16. The detection method according to claim 1, furthercomprising measuring a number of the biological micro particles that areliving cells based on at least the detected radiation.